1. Field of the Invention
The present invention relates to a biosensor that can easily quantify a specific component in a sample liquid with accuracy and speed, and a method for producing the same, and more particularly to a biosensor for quantifying a specific component in a sample liquid by reducing electron acceptors using electrons generated in the reaction of the specific component in the sample liquid to enzyme that specifically reacts to the component, and then by electrochemically measuring the reduced amount of electron acceptors, and a method for producing the same.
2. Description of the Prior Art
Various types of biosensors utilizing specific catalyses of enzyme have been recently developed. A saccharide biosensor will be described as an example of such biosensors as follows:
The optical rotation method, the colorimetric method, the reductimetry method and other methods using different kinds of chromatographies have been developed as method for quantitative analysis of saccharides. However, none of these methods can provide high accuracy due to the relatively low specificity against saccharides. Additionally, the optical rotation method is easy to operate but is largely influenced by the operating temperature. Therefore, it is not appropriate for common use at home and the like.
The saccharides contained in fruit are generally assessed as saccharine degrees. A refractometer of the light refraction system is often used for quantifying the saccharine degree. This refractometer functions by utilizing change of the light refractive index caused by liquid concentration. Therefore, the refractometer of the light refraction system is influenced by all the components dissolved in the sample liquid, for example, by organic acid such as citric acid or malic acid contained in fruit juice in a large amount when a saccharide in fruit is quantified. Thus, accurate quantification by this refractometer is impossible.
A glucose sensor will now be described as an example of a biosensor used in a clinical field.
A conventional method for quantifiying glucose contained in blood is to centrifuge blood taken from a patient and then to measure the thus obtained blood plasma. This method requires a lot of time as well as labor. Therefore, a sensor that can directly measure glucose in blood obtained from a patient has been desired.
A sensor similar to a test paper for urinalysis has been developed as a simple glucose sensor. This glucose sensor comprises a support in a stick shape and a holder fixed to the support. The holder includes an enzyme reacting only to glucose and a dye the color of which is changed by reacting with a production of the enzyme reaction. Blood is dropped on the support of the glucose sensor and the change of the dye after a predetermined period of time of the dropping is visually observed or optically measured, whereby the content of glucose in the blood can be measured. However, the quantifying method using this glucose sensor has low accuracy due to the interference by the colored materials in the blood.
Japanese Laid-Open Patent Publication No. 1-114747 discloses the following glucose sensor with high accuracy as a method for quantifying a specific component in a sample liquid from a living body such as blood without diluting or stirring the sample liquid:
As shown in FIG. 7 this glucose sensor comprises an electrical insulating substrate 51, an electrode system 54 including a working electrode 52 and a counter electrode 53 formed on the insulating substrate 51 by screen printing, an electrical insulating layer 55 formed on the insulating substrate 51, an adhesive structure 56 provided on the electrical insulating layer 55, a filtration layer 57 supported by the adhesive structure 56, a holding frame 58 provided on the filtration layer 57, and an electron acceptor holding layer 59, an enzyme holding layer 60, a buffering salt holding layer 61 and an expansion layer 62 which are supported by the holding frame 58. A space 63 for containing a sample liquid is formed between the electrode system 54 and the filtration layer 57.
The filtration layer 57 is made from a porous polycarbonate film. The electron acceptor holding layer 59, the enzyme holding layer 60 and the buffering salt holding layer 61 use porous cellulose as supports.
The operation of such a glucose sensor is as follows: The sample liquid dropped on the expansion layer 62 is adjusted to pH that provides the most stable enzyme activity by the action of the buffering salt in the buffering salt holding layer 61. Then, in the enzyme holding layer 60, the glucose oxidase in the enzyme holding layer 60 and the glucose in the sample liquid specifically react to each other. The potassium ferricyanide in the electron acceptor holding layer 59 is then reduced to become potassium ferrocyanide by electrons generated in the above reaction. The amount of the potassium ferrocyanide generated at this time is in proportion to the glucose concentration in the sample liquid. Next, materials with larger molecular weight such as protein are filtered in the filtration layer 57, and the filtered liquid drops in the space 63 above the electrode system 54. Thus the amount of potassium ferrocyanide in the liquid can be measured by measuring the oxidation current of the liquid by the electrode system 54, thereby measuring the glucose concentration.
In the conventional glucose sensor with the above-mentioned structure, frothing may remain in the space due to the inconstant flow of the liquid in the space 63, which influences the measured value of the glucose concentration.
Furthermore, since the buffering salt holding layer 61 is in contact with the enzyme holding layer 60, the buffering salt and the enzyme are mixed on the interface between the two layers when the glucose sensor absorbed moisture, thereby deteriorating the enzyme activity by the chemical interaction. As a result, the glucose sensor of this type is hard to store in a stable condition.
Moreover, since insoluble porous materials are used as supports of the filtration layer 57 and each of the holding layers 59, 60, and 61 in the conventional glucose sensor, the sample liquid supplied to the glucose sensor is required to pass through each of the porous materials before reaching the electrode system 54. Therefore, the sensor has disadvantages that it may take longer time to obtain the reaction and/or that the response values may be variable due to the variable reaction time. Additionally, the glucose sensor has so many steps in its production, including such a complicated step as to assemble a plurality of porous materials, that it is difficult to produce it at a low cost.